Carbon Assimiilation and Translocation in Soybean Leaves at Different Stages of Development

Carbon assimilation, translocation, and associated biochemical characteristics of the second trifoiolate leaf (numbered acropetally) of chambergrown soybean, Glycine max (L.) Meff., plants were studied at selected stages of leaf development during the period from 10 to 25 days postemergence. Leaves of uniform age were selected on the basis of leaf plastochron index (LPI). The test leaf reached ful expansion (A..) and maximum CO2 exchange rates on a leaf area basis at 17 days postemergence (LPI 4.1). Maximum carbon exchange rates per unit dry weight of lamina were attained several days earlier and declined as specific leaf weight increased. Chlorophyl and soluble protein continued to increase beyond the attainment of A.., but were not accompanied by further increases in photosynthetic rates. Much of the fixed carbon in leaves is partitioned between starch and sucrose. Starch content of leaves as a percentage of dry weight at the end of an 11-hour photoperiod was taken as an indication of the potential energy reserve accumulated by the leaf. Starch levels were the same regardless of leaf age during the period from 0.3 Ami. to 7 days after attaining A... Respiratory and synthetic activity of leaves decreased considerably during the same period, suggesting that starch accumulation is not entirely controlled by the energy demands of the leaf. Sucrose content increased steadily during leaf expansion and was accompanied by corresponding increases in sucrose phosphate synthetase (EC 2.4.1.14) activity and translocation rates. Sucrose phosphate synthetase may have an important regulatory role in photosynthate partitioning and translocation. The growth of green plants depends not only upon photosynthesis, but also upon the translocation of photosynthates from sites of carbon fixation in differentiated photosynthetic tissue to sites of storage or utilization where growth and differentiation occur. Photosynthesis and translocation rates in expanding leaves reach maxima as leaf expansion ceases, and decline shortly thereafter (8, 13). The efficiency of the leaf as an assimilatory organ depends upon a large array of biochemical and physiological processes which exist in a dynamic relationship with leafontogeny. The present study is part of an effort to identify physiological and biochemical processes which restrict the photosynthetic efficiency of leaves. Our approach was to determine the rates of carbon assimilation and translocation at precisely identified stages of leaf development. At each stage of development, specific biochemical components of the assimilatory process were characterized. A comparison of the changes in these components in relation to the changes in the over-all assimilatory activity during leaf development provides a basis for identifying those components which may restrict the efficiency ofcarbon assimilation in soybean leaves. 54 MATERIALS AND METHODS Plant Material. Soybean, Glycine max (L.) Merr. cv. Amsoy 71, plants were grown from uninoculated seed in 15-cm plastic pots containing Vermiculite. Four days after emergence, seedlings were thinned to one plant/pot. Incandescent and cool-white fluorescent lamps supplied a photosynthetic photon flux density (400-700 nm) of 500 ILE m2sec ' at pot height during a 14-hr photoperiod. Temperature and RH were maintained at constant levels of 27 + I C and 60 + 2%, respectively. Horizontal temperature variation within the chamber was less than 2 C. The plants received an excess of a nutrient solution developed by F. W. Snyder of this laboratory; it contains the following salts (mM concentration): Ca(NO3)2, 4; KCI, 2.5; KH2PO4, 1; KNO3, 3; K2SO4, 1; MgSO4, 3.5; NH4H2PO4, 2.5; and the following micronutrients (uM concentration): H3BO3, 13.7; CUSO4, 0.16; MnSO4, 4.5; (NH4)6Mo7024, 0.07; ZnSO4, 0.34; metallic Fe, 107.4, as Sequestrene 330 Fe powder formulation (CIBA-GEIGY Corp., Greensboro, N.C.).' All observations were made on the second trifoliolate leaf (T2) numbered acropetally at six stages of development from 11 to 25 days postemergence. In order to reduce ontogenetic variability, plants were selected for each stage on the basis of plastochron index (Pl)2 (7). The PI system was devised by Erickson and Michelini (7) to provide a precise numerical indication of the stage of development of a vegetative shoot and individual leaves thereon. Briefly, the PI of soybean was calculated as follows: the parameter chosen to indicate leaf size was the midvein length of the terminal leaflet. A reference length of 20 mm was arbitrarily selected. Then, the node number (n) having the youngest leaf with a reference length exceeding 20 mm was determined by counting acropetally from the cotyledonary node. The midvein length of this leaf (Ln), and that of the leaf at the adjacent node above (L+,I) were measured and the PI of the plant calculated from the following relationship: PI = n + log L4-l log L log L4+, The leaf plastochron index (LPI) of a leaf at any given node, n, equals PI minus n. Carbon Assimilation and Translocation. CO2 exchange rate (CER) for the attached T2 leaves was measured under growth ' Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products or vendors that may also be suitable. 2 Abbreviations: F6P: fructose 6-phosphate; UDPG: uridine diphosphate glucose; ADPG: adenine diphosphate glucose; SPS: sucrose phosphate synthetase; SPP: sucrose phosphate phosphatase; CERA: carbon dioxide exchange 'rate-leaf area basis; CERw: carbon dioxide exchange rate-leaf dry weight basis; LPI: leaf plastochron index; PGA: 3-phosphoglyceric acid; PI: plastochron index; SLW: specific leaf weight. www.plantphysiol.org on July 22, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. CARBON ASSIMILATION IN SOYBEAN conditions in acrylic plastic chambers representing an adaptation of the air-seal technique (28). Four leaf chambers were connected in parallel fashion into a flow-through IR gas analysis system. The CER for each of the four T2 leaves was measured hourly for 10 hr following I hr of acclimation at the beginning of the light period. In order to express CER and translocation rates in the same units, mg of CO2 was converted to mg of CH20 since a large proportion of leaf organic matter is represented by this empirical formula; thus, CERA = CER x 0.68, where 0.68 represents the molar ratio of the two forms of carbon. The CER is expressed on a leaf area basis as mg of CH2O dm2hr-' (CERA) and on a leaf dry wt basis as Tg of CH20 g-'hr-' (CERw); CERw = CERA X I/SLW (mg dm-f). During the last two stages of development, neighboring leaves were reoriented to maintain leaf T2 at the same photosynthetic photon flux density as in previous stages of development. Dark respiration rates were determined from CO2 exchange rates during the 30 min of darkness before the start of the photoperiod. Three leaf discs, each having an area of 0.52 cm2, were removed from T2 of each of seven plants at the beginning and at the end of the 10-hr period. The discs were placed in small envelopes, immediately frozen in liquid N2, and lyophilized. Dry wt accumulation (mg dm hr-') was determined from the dried discs. Mass carbon translocation rates were estimated from the difference between total carbon fixed and the dry wt accumulation according to the method of Terry and Mortimer (22). Validity of their method was checked by comparing resultant translocation rates with those determined from export of '4C from '4CO2-fed leaves at comparable stages of leaf development according to the method of Thorne and Koller (23). Following the 10-hr test period, leaf areas of T2 leaves were determined (LI-COR model LI-3000 portable area meter, Lambda Inst., Inc., Lincoln, Neb.), the leaf blades excised, frozen in liquid N2, lyophilized, and stored at -20 C for chemical analyses and enzyme assays. Chemical Analyses. Finely ground 200-mg samples of lyophilized leaves were extracted twice in boiling 80o ethanol, the extracts combined, and the ethanol removed by evaporation. Sucrose concentration of the aqueous extracts was determined according to the method of van Handel (27). Starch concentration was estimated by titration of reducing sugars following enzymic digestion of the remaining solid residue (21). Sucrose and starch content of the leaves were expressed as a percentage of dry wt. Chi content was determined according to the method of Schmid (18). Extraction and Assay of Enzymes. Samples of lyophilized leaf blades weighing 100 mg were ground in a mortar with sand and 5 ml of extraction medium containing 0.1 M HEPES buffer (pH 7) and 20 mM mercaptoethanol. The extract was centrifuged at 35,000g for 15 min and the supernatant passed through a column (I x 15 cm) of Sephadex G-25 (coarse) equilibrated with 5 mm HEPES buffer (pH 7) containing Imm mercaptoethanol. Aliquots (0.1 ml) were removed from the supernatant and from the Sephadex eluant for protein analysis (14). All steps were performed at